1. Field of the Invention
The present invention relates to a method for the detection of an analyte in a sample, and more particularly concerns a method employing an enzyme immunoassay for measuring the concentration of an analyte in a sample by indirect colorimetric detection.
2. Background Description
Assays for the colorimetric detection of analytes are common and well-known. In particular, enzyme immunoassays (EIA) and enzyme-linked immunosorbent assays (ELISA) are employed for the colorimetric detection of an antigen (analyte) present in a test sample. In these enzyme immunoassay methods, antibodies specific to the test antigen or analyte are bound to a solid phase which is usually a clear or transparent plastic tube. In many, but not all, of these enzyme immunoassays, a sandwich formation occurs between the solid phase antibody, the antigen and the enzyme-labeled antibody such that the amount of enzyme-labeled antibody bound to the solid phase is directly proportional to the antigen concentration in the sample. Excess, unreacted reagents are normally washed from the tube, and substrate is added to effect the enzyme reaction.
In addition to sandwich formation, which typically occurs with protein antigens, it is possible to perform competitive assays. A limiting amount of antibody is put on the solid phase, and both sample and an enzyme-labeled hapten are simultaneously incubated. When no analyte is present in the sample, the enzyme-labeled hapten binds maximally to the solid phase. As the amount of analyte in the sample increases, it competes with the enzyme-labeled hapten for binding to the solid phase such that the amount of enzyme-labeled hapten binding is inversely proportional to the analyte concentration. Competitive assays may be performed for both haptens and protein analytes, but only protein analytes are amenable to a sandwich-type assay.
In colorimetric detection techniques, the substrate may include a chromogenic substance which is responsive to the enzyme so that the chromogenic substance is activated. As a result, sufficient color is produced in the liquid solution within the tube which may be detected. Assays of this nature are frequently completed by measuring the color production within the tube by use of a spectrophotometer. In the event that fluorochromes are used as the substances to be detected, fluorescence microscopes as well as fluorometers may be used to perform the assay methods. An enzyme immunoassay using a tetramethylbenzidine (TMB) as the chromogen in the colorimetric detection of an antigen is described in U.S. Pat. No. 4,503,143.
In using a spectrophotometer for the colorimetric detection of an analyte in a sample, there are a number of factors which may affect the detection of light associated with the chromogenic reaction. For instance, the light source for providing an incident beam of light into the tube containing the sample to be tested is frequently a lamp, for instance, a tungsten lamp, such as that described in U.S. Pat. No. 4,516,856. It is not uncommon to have variations in lamp output which may affect the light detected in a spectrophotometer or light collected in a photomultiplier for detecting fluorescence emissions. Positioning of the tube containing the sample to be tested may be different from test to test, also having an affect on the test results. Other factors such as light path length and optical quality of the tube may produce differences from test to test which may affect the assay results. It is believed that a technique which corrects or obviates the aforementioned variables in colorimetric detection methods would be most desirable in improving the accuracy, reproducibility and reliability of these test procedures.
Improvements in fluorometric assays have been described in U.S. Pat No. 4,495,293. However, the object of the just-mentioned patent is to provide improvements in the fluorescent intensity of the emitted light which is related to the concentration of the ligand. Another technique for the assays of macromolecules by immunonephelometry is described by DeGrella et al. in "A Nephelometry System for the Abbott TDx.sup.198 , " Clin. Chem. 31/9, 1474-1477 (1985). A nephelometric method for monitoring chromogenic reactions is described in this publication, which method relies on scattered energy attenuation to measure serum proteins. There are no teachings or suggestions, however, in the DeGrella et al. article which would be useful in correcting for the variables, as set forth above, which affect the assay measurements.
Accordingly, improvements are still being sought in assays relying on the colorimetric detection of the analytes of interest. The present invention is directed to such an improvement.